PROJECT SUMMARY Copper (Cu) is essential for the growth, development, and normal function of human organisms. The loss of Cu homeostasis is associated with a broad spectrum of pathologies including Menkes disease, Wilson disease, MEDNIK syndrome, and others. So far, the studies of human Cu homeostasis have focused primarily on the function and regulation of Cu transporters and small Cu carriers. With this program of study, we will begin to learn how mammalian Cu transporters and their regulators work together to modulate nutrient transport in the intestine. The small intestine is responsible for the absorption of all essential nutrients. We discovered that the availability of Cu in enterocytes strongly influenced the abundance of chylomicrons, the primary carriers of dietary fat. We have also identified the fat-responsive protein ANKRD9 as a regulator of Cu transport and hypothesize that ANKRD9 is a molecular integrator of the pathways involved in the intestinal fat and Cu transport. The proposed program of studies will test the central hypothesis that the Cu homeostasis and lipid (fat) metabolism in enterocytes are functionally linked and co-regulated. Studies under Specific Aim 1 will characterize the mechanism of ATP7B regulation, investigate how the intestinal Cu storage compartments are formed, and determine how ATP7B modulates the distribution of Cu between the major cellular compartments. Specific Aim 2 will determine how Cu and dietary fat affect each other transport in neterocytes and elucidate the mechanism behind the Cu-dependent formation of chylomicrons. Specific Aim 3 will characterize the function of ANKRD9 in the small intestine. The studies will open a new chapter in understanding of intestinal Cu physiology, contribute to better understanding of human disorders associated with Cu misbalance and, ultimately, help to design better treatments for these disorders.